Anti-cryptosporidium parvum preparations

ABSTRACT

Compositions and methods useful for conferring passive or active immunity to the parasite,  C. parvum . A high molecular weight glycoprotein antigen isolated from  C. parvum , capable of binding the mAb 3E2, was shown to harbor an epitope critical for triggering the neutralizing CSP-like reaction in the parasite. Antibodies targeted against the critical epitope were shown to possess neutralizing activity, and could be combined with other anti- C. parvum  monoclonal antibodies and administered to an animal to confer passive immunity. Immunogenic compositions including the purified antigen are disclosed for use in stimulating an active immune response against  C. parvum.

RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.09/557,324, filed Apr. 25, 2000, now allowed, which is a Continuation ofU.S. application Ser. No. 08/828,943, filed Mar. 27, 1997, now U.S. Pat.No. 6,110,463, issued on Apr. 29, 2000, which claims priority to U.S.Provisional Application Ser. No. 60/014,410, filed Mar. 29, 1996, and toU.S. Provisional Application Ser. No. 60/021,465, filed Jul. 10, 1996.

GOVERNMENT INTEREST IN THE INVENTION

Certain aspects of the invention disclosed herein were made with UnitedStates government support under Public Health Service (NIH), GovernmentContract No. U01 A1 30223; USDA Special Grant, Contract No.89-34116-4550; and USDA NRICGP, Contract No. 94-37204-0496. The UnitedStates government has certain rights in these aspects of the invention.

FIELD OF THE INVENTION

The present invention relates to preparations useful for conferringpassive or active immunity to the parasite, Cryptosporidium parvum. Morespecifically, the invention relates to a purified glycoproteinconstituent of the parasite; use of the glycoprotein in an immunogeniccomposition; and monoclonal antibodies that bind a particular epitopedisposed on the glycoprotein.

BACKGROUND OF THE INVENTION

Cryptosporidium parvum is a coccidian parasite that causes intestinaldisease in humans as well as economically important food animalsincluding calves, lambs, and goat kids. Healthy, immunocompetent adulthumans can be infected, but cryptosporidiosis is particularly seriouswhen it occurs in immunodeficient individuals, including neonates, andthose who are immunocompromised as a result of medical treatment, orbecause of other disease, such as infection by human immunodeficiencyvirus (HIV).

Among domestic animals, cryptosporidiosis is most frequently reported incalves. The ubiquity of C. parvum in dairy and beef operationsthroughout the U.S. and its importance as a cause of calf diarrhea arewell documented. For example, Anderson et al. in Vet. Med. Sm. Anim.Clin. (June 1981) described well-managed, closed-herd dairies in whichC. parvum-related morbidity in 1-2 week old calves approached 100%. Weconservatively estimate that the combined treatment costs and decreasedproduction losses incurred by the U.S. cattle industry due tocryptosporidiosis alone now exceed $50,000,000 each year.

C. parvum infection begins when sporozoites released from ingestedoocysts invade intestinal epithelial cells. Following attachment of theanterior pole of sporozoites to intestinal epithelium, invasion isassociated with host cell membrane evagination around the sporozoite andparasitophorous vacuole formation. Vacuole formation in the apicalcomplex of invading sporozoites is thought to represent discharge ofinvasion mediators from apical organelles. Following invasion, a feederorganelle forms between the parasite and the host cell cytoplasm andincreases the interface surface area markedly. This organelle mayfunction in transport of materials between the host cell and developingtrophozoite. Two stages of merogony follow trophozoite development. Type1 merozoites undergo cyclic replication before developing into type 2merozoites. Type 2 merozoites subsequently give rise to sexual stages.Fertilization follows and results in the production of oocysts whichsporulate at the time of passage in feces. Autoinfective sporozoite andmerozoite loops in the life cycle may perpetuate infection inimmunocompromised hosts.

Since the first cases of human cryptosporidiosis were reported in 1976,Cryptosporidium has become recognized as a common cause of diarrhea ininternational travelers, children in day-care centers, livestockhandlers, and patients with AIDS or other immune deficiency disorders.Among several recent studies that have addressed the prevalence of C.parvum infection in AIDS patients with diarrhea, one study identifiedCryptosporidium as the most common enteropathogen in diarrheic AIDSpatients (Laughon et al. Gastroenterol. 94:984 (1988)). Dissemination toextraintestinal sites such as the esophagus, lungs, pancreas and liverhas also been shown to occur in immune deficient patients (Soave et al.Rev. Inf. Dis. 8:1012 (1986); Ungar et al. “Cryptosporidiosis in Humans”pp. 67-75, in J P Dubey, C A Speer and R Fayer (eds.), Cryptosporidiosisof Man and Animals, CRC Press (1990)).

Unlike the other major causes of diarrhea, including infection by E.coli, rotavirus and coronavirus, there are no effective control measuresavailable for cryptosporidiosis. Despite the evaluation of more than 90drugs, none has been of consistent value and no immunization regimen ispresently available to protect against infection by C. parvum. Controlof C. parvum infection therefore depends on achieving an adequate immuneresponse. An index of adequate response comprises resistance toreinfection following recovery and short-term disease in immunocompetenthosts. The disease may persist however in immunodeficient hosts.

While cell-mediated immunity is important to naturally occurringresistance to many coccidial species, the evidence suggests thatantibody responses can also be manipulated to control infection withsporozoan parasites. For example, hyperimmune bovine serum or colostralwhey against whole C. parvum neutralized sporozoite infectivity andpartially protected mice and calves against oocyst challenge.Additionally, oral administration of hyperimmune bovine colostrum topersistently infected immunodeficient patients was followed by cessationof diarrhea and oocyst shedding. Further, monoclonal antibodies (mAbs)reactive with C. parvum sporozoite and merozoite surface epitopesneutralized their infectivity and partially protected mice againstoocyst challenge.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided amonoclonal antibody having the epitope binding specificity of antibody3E2. In one embodiment, the monoclonal antibody and antibody 3E2 competewith each other for binding to an antigen that is present in apreparation solubilized C. parvum sporozoites. This monoclonal antibody,which competes with antibody 3E2 for antigen binding, stimulates aCSP-like reaction after contacting C. parvum sporozoites. According to adifferent embodiment, the monoclonal antibody can have an IgM isotype.In a particular case, the monoclonal antibody is antibody 3E2. Theinvention also provides a hybridoma that secretes the monoclonalantibody 3E2.

According to a second aspect of the invention there is provided apharmaceutical composition for administration to a mammal, comprising amonoclonal antibody secreted by hybridoma 3E2 and a pharmaceuticallyacceptable carrier. The invented composition also can include at leastone monoclonal antibody other than the monoclonal antibody secreted byhybridoma 3E2. In a preferred embodiment, this other monoclonal antibodyhas an epitope binding specificity different from the bindingspecificity of the monoclonal antibody secreted by hybridoma 3E2. Thecarrier of the pharmaceutical composition can include at least onestabilizing agent which may be a protease inhibitor, a carrier proteinor a pH buffering agent. In one embodiment, the carrier optionallycomprises colostrum, for example, bovine colostrum.

A third aspect of the invention relates to a method of providing to amammal passive immunity against C. parvum infection, comprising the stepof administering to the mammal a composition comprising antibody 3E2,thereby providing passive immunity. In one embodiment, the compositionadministered to the mammal comprises a C. parvum neutralizing amount ofmonoclonal antibody 3E2. According to a different embodiment, theadministered composition includes at least one monoclonal antibody otherthan antibody 3E2 that specifically binds a C. parvum antigen. In apreferred method the composition comprising antibody 3E2 is administeredorally in the administering step. In another preferred embodiment of themethod, the mammal is a human and in a particularly preferred embodimentthe mammal is an immunocompromised human.

A fourth aspect of the invention relates to an isolatedcircumsporozoite-like antigen of C. parvum which includes a glycoproteinhaving a molecular weight of 1,400 kDa and which harbors an epitopespecifically recognizable by monoclonal antibody 3E2. The isolatedantigen can be isolated by a method that includes centrifugation, moreparticularly, density gradient centrifugation. Alternatively, theisolated antigen can be isolated by methods that involveimmunoprecipitation, isoelectric focusing or preparative polyacrylamidegel electrophoresis.

A fifth aspect of the invention relates to an immunogenic compositionwhich includes: (a) a substantially purified C. parvum antigenspecifically recognizable by monoclonal antibody 3E2; and (b) apharmaceutically acceptable carrier. In one embodiment, the C. parvumantigen is a glycoprotein with a molecular weight of approximately 1,400KDa. In another embodiment, the carrier of the immunogenic compositioncan include an adjuvant.

A sixth aspect of the invention relates to a method of neutralizing C.parvum infection in a mammal, where the method includes the step ofadministering to the mammal a composition which includes an antibodyhaving binding specificity for an epitope specifically recognizable bymAb 3E2. The administered composition can include monoclonal antibody3E2 itself, and may include a C. parvum neutralizing amount ofmonoclonal antibody 3E2. Alternatively, the administered composition caninclude at least one monoclonal antibody other than monoclonal antibody3E2. The administered composition can be administered orally.

Still another aspect of the invention relates to a method of stimulatingan anti-C. parvum immune response in an animal. Practice of the inventedmethod involves first obtaining an immunogenic composition that includesa purified C. parvum antigen dispersed in a pharmaceutically acceptablecarrier, where the antigen is specifically recognizable by mAb 3E2, andthen administering the composition to the animal according to avaccination protocol. The immunogenic composition used in the inventedmethod optionally can include an adjuvant.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As disclosed herein, the 3E2 monoclonal antibody (mAb) hasbinding-specificity for a C. parvum surface and apical glycoproteincomplex of sporozoites and merozoites, and efficiently neutralizes C.parvum infection in vivo. Interestingly, the epitope recognized by themAb 3E2 exhibits properties consistent with acarbohydrate/carbohydrate-dependent structure. More particularly, thetarget epitope, immobilized in a Western blotting format, was sensitiveto degradation by glycosidase and degradation by periodate treatment,but resistant to protease digestion in a dot-blotting format. Thus, itis highly unlikely that the epitope could be expressed in recombinantform in a procaryotic host, for example. As will be apparent from thefollowing disclosure, the production and isolation of the 3E2 hybridomawas made possible by the use of purified native C. parvum antigens asimmunogens.

The present invention is useful for treating and preventingcryptosporidiosis in all mammals, including human neonates, and also inimmunodeficient and certain immunocompetent children and adults. Moreparticularly, the invented composition comprising the mAb 3E2 can beadministered to young ruminants, for example, calves, goats and sheep.The invention is useful in these applications because young domesticruminants serving as food animals are typically housed under conditionswhere exposure to C. parvum is unavoidable and typically do not possessfunctionally mature immune systems capable of fighting off infection atthe time of exposure to the parasite. Another application of theinvention relates to the control of cryptosporidiosis in humans havingacquired or congenital immunodeficiencies, including individuals havingacquired immunodeficiency syndrome (AIDS) resulting from infection withthe human immunodeficiency virus (HIV).

Therapeutic or prophylactic compositions falling within the scope of theinvention include preparations containing the mAb 3E2, optionallycontaining stabilizing agents including protease inhibitors, carrierproteins and pH buffering agents. A convenient source of agents known tostabilize antibodies, particularly when administered by oral route, iscolostrum. Colostrum naturally contains antibodies which can, forexample, be passed to a neonate. Thus, contemplated formulations fordelivery of mAb 3E2 may involve dispersion of the mAb into colostrumprior to administration to a recipient mammal. Notably, experimentalresults presented herein proved that ascites preparations could beadministered orally with strong retention of C. parvum neutralizingactivity characteristic of mAb 3E2. This confirmed the utility of mAb3E2 as an agent having powerful neutralizing activity directed againstthe parasite, C. parvum.

As disclosed herein, we have employed competition binding assays toidentify mAbs having binding specificities for the same, similar oroverlapping epitopes. Results of testing using these assays revealedthat mAbs which neutralize C. parvum infection in vivo and which elicitthe CSP-like reaction upon contacting sporozoites all have bindingspecificities that overlap the binding specificity of mAb 3E2. In viewof the identity between neutralizing mAbs having the ability to elicitthe CSP-like reaction and those having the ability to compete with mAb3E2 for antigen binding, we concluded that identification of mAbs thatcompete with mAb 3E2 for antigen binding represents a method ofidentifying mAbs that can stimulate the CSP-like reaction in C. parvum.Moreover, our results support that any mAb that specifically competeswith mAb 3E2 for antigen binding will also have neutralizing activityand be capable of stimulating the CSP-like reaction.

Another aspect of the invention relates to a method of stimulating ananti-C. parvum immune response in an animal using as an immnunogen apurified glycoprotein constituent of C. parvum, wherein the constituentis characterized as a target for binding by mAb 3E2. Immunization withcharacterized molecules known to be the antigenic targets of aneutralizing humoral immune response, advantageously focuses the immuneresponse against critical epitopes rather than against potentiallyirrelevant epitopes otherwise found in whole organism preparations. Asdescribed below, we have now defined critical epitopes disposed on thesurface of C. parvum to which an antibody response must be directed inorder to optimize immunoprotection against infection by this organism.

The hybridoma cell line secreting mAb 3E2-A8-B2 (3E2) has been depositedon Mar. 28, 1996 with the American Type Culture Collection Manassas, Va.in compliance with the procedures specified for the deposit ofbiological materials under the Budapest Treaty. The deposit has beenassigned accession number ATCC HB 12075.

The starting point for the development of the invention involved thepurification of particular native antigens from C. parvum sporozoites.The C. parvum isolate used in the procedures described herein wasobtained from H. Moon (National Animal Disease Center, Ames, Iowa). TheC4A1 mAb, described in detail by Mead et al. (J. Parasit. 74:135 (1988))and Arrowood et al. (Infect. Immun. 57:2283 (1989)), was used in thefollowing procedure as an immunoaffinity purification reagent.Significantly, the purification protocol described below advantageouslyallowed the isolation of C. parvum protein constituents havingposttranslational modifications that would have been absent from, forexample, recombinant antigens produced in a heterologous host. Generalmethods useful in immunoaffinity purification procedures can be found inMonoclonal Antibodies: Principles and Practice, 2nd ed. J. W. Goding,Academic Press, London (1986) pp. 219-227, the disclosure of which ishereby incorporated by reference.

Example 1 describes one procedure useful for isolating antigenconstituents of C. parvum.

EXAMPLE 1 Isolation of C. parvum Antigens

The C4A1 mAb was purified from ascites fluid by an initial mixed moderesin cation exchange and affinity chromatography step and subsequentlypurified by HPLC to give an antibody preparation that was 96% pure IgM.These standard procedures were performed by the commercial laboratoryservice, TSD Bioservices (New York). Purified C4A1 was coupled tocyanogen bromide-activated sepharose 4B (Pharmacia Inc., Piscataway,N.J.) according to manufacturer's instructions. The derivatized matrixwas then loaded into a column and optimal binding and elution conditionsuseful for isolating immobilized antigen were determined. Preparativelevel purification experiments were performed using C. parvumsporozoites that had been solubilized in lysis buffer consisting of 50mM Tris HCl, 5 mM EDTA, 5 mM iodoacetarnide, 0.1 mM N-a-p-tosyl-L-lysylchloromethyl ketone (TLCK), 1 mM phenylmethanesulfonyl fluoride (PMSF)and 1% (wt/vol) octyl glucoside. Solubilized material was bound to theC4A1-coupled matrix, washed extensively and immobilized material elutedwith 0.1 M glycine (pH 3.3).

Samples representing solubilized oocysts, solubilized sporozoites andimmunoaffinity purified material were separated on a 10-20%polyacrylamide gel under reducing and denaturing conditions and theprotein bands visualized by silver staining. The stained gel showedcomplex banding patterns representing more than approximately 100-150protein species in the size range extending from approximately 21.5 kDato greater than 200 kDa in the lanes corresponding to the crudesolubilized preparations. In contrast, the lane representingimmunoaffinity purified material gave a simpler pattern consisting ofapproximately 17 distinct protein bands. The stained bands wereconcentrated in the 25 to approximately 230 kDa size range. Theseresults confirmed that the immunoaffinity purification proceduredescribed above was useful for purifying native sporozoite constituents.

Western blotting using the C4A1 mAb as a probe was performed essentiallyaccording to the method described by Riggs et al. in Infect. Immun.62:1927 (1994). In this procedure, samples of solubilized C. parvumsporozoites and immunoaffinity purified material wereelectrophoretically separated and Western blotted in parallel.Affinity-purified alkaline phosphatase conjugated goat anti-mouse IgMwas used to detect binding of the C4A1 mAb following substrate addition.Results of the procedure indicated a correspondence between bandsobserved by SDS-PAGE and silver staining and immunoreactive proteinsdetectable by C4A1 staining in Western blots. Further, immunoreactivebands in immunoaffinity purified antigens comigrated with bands ofsimilar molecular weight derived from whole organisms. This stronglysuggested that the antigenic target of the C4A1 mAb was not a singlespecies, but a small number of antigens that shared the same epitope. Asa result of this size heterogeneity, the antigen recognized by the C4A1mAb has been referred to as the “GP25-200” complex.

The immunoaffinity purified GP25-200 complex isolated according to themethod described above was subsequently used as an immunogen in a steptoward producing an expanded panel of hybridomas. General methods usefulin the production of mAbs can be found in Monoclonal Antibodies:Principles and Practice, 2nd ed. J. W. Goding, Academic Press, London(1986) pp. 59-93, the disclosure of which is hereby incorporated byreference.

Example 2 describes the procedures used for immunizing mice with theimmunoaffinity purified GP25-200 complex and producingantibody-secreting hybridomas.

EXAMPLE 2 Immunization of Mice with Purified GP25-200 Antigen andPreparation of Hybridomas

Adult female BALB/c mice (Harlan Sprague Dawley, Indianapolis, Ind.)were immunized with 2 μg of immunoaffinity purified GP25-200 antigenincorporated in monophosphoryl lipid A trehalose dimycolate adjuvant(R-700, Ribi, Hamilton, Mont.) by intraperitoneal (i.p.) andsubcutaneous (s.c.) administration. A second injection of 1 μg GP25-200in adjuvant was given 7 weeks later by the same routes. Four weekslater, a third injection of 1 μg GP25-200 in adjuvant was given s.c. Afinal intravenous (i.v.) injection of 1.5 μg GP25-200 in phosphatebuffered saline was given 5 weeks later. Three days following i.v.injection, spleens were removed and single cell suspensions prepared forfusion with SP2/0 myeloma cells. Cell fusions using SP2/0 myeloma cellsand cloning by limiting dilution were performed as described by McGuireet al. in Am. J. Vet. Res. 44:1284 (1983).

Preliminary indirect immunofluorescence assays using heat-fixedsporozoites as antigenic targets were used to identify hybridomas havingspecificity for GP25-200. Results from this preliminary screeningindicated that 112 of the hybridomas were positive for sporozoitebinding. These hybridomas were found to have heterogeneous indirectimmunofluorescence patterns that included apical reactivity, surfacemembrane reactivity in a multifocal or diffuse pattern and/or multifocalinternal binding.

Hybridoma supernatants that gave positive staining results in thepreliminary assay were then screened for surface-reactive anti-GP25-200antibody by the live indirect immunofluorescence assays described byRiggs et al. in Infect. Immun. 55:2081 (1987) and Mishell et al. inSelected Methods in Cellular Immunology, B. B. Mishell and S. M. Shiigi,eds. W.H. Freeman and Company, San Franscisco, Calif. p.19 (1980). Thedisclosures of these two citations are hereby incorporated by reference.Fluorescein-conjugated goat anti-mouse Ig (IgA, IgG, IgM) secondantibody (Cooper Biomedical, Malvern, Pa.) was used to detect binding ofanti-GP25-200 antibody. mAb isotypes and concentrations were determinedby immunodiffusion according to the method of Johnstone et al. inImmunochemistry in Practice, A. Johnstone and R. Thorpe, eds. BlackwellScientific Publications, Boston, p. 120 (1982).

Example 3 describes the method used to screen hybridoma supernatants toidentify mAbs specific for the GP25-200 antigen.

EXAMPLE 3 Indirect Immunofluorescence Assay to Identify mAbs Specificfor GP25-200

Hybridoma culture supernatants were screened for surface-reactiveanti-GP25-200 antibody by live indirect immunofluorescence according tothe method described by Riggs et al. in Infect. Immun. 55:2081 (1987)and Mishell et al. in Selected Methods in Cellular Immunology, B. B.Michell and S. M. Shiigi, eds. W.H. Freeman and Company, San Franscisco,Calif. p.19 (1980), the disclosures of which are hereby incorporated byreference. All steps were performed at 4° C. by using viable sporozoites(5×10⁷/ml) in PBS with 0.5% (wt/vol) bovine serum albumin (PBS-BSA;Sigma Chemical Co., St Louis, Mo.). The assay was performed in 96-wellV-bottom microtiter plates (Flow Laboratories). All dilutions were madewith PBS-BSA. A total of 50 μl of hybridoma supernatant and 50 μl ofsporozoite preparation per well were incubated for 30 minutes and thenwashed four times with 200 μl of PBS-BSA per well (3,500×g). A total of50 μof fluorescein-conjugated goat anti-mouse Ig (IgA, IgG, IgM) secondantibody (Cooper Biomedical, Malvern, Pa.) was used to detect binding ofanti-GP25-200 antibody. mAb isotypes and concentrations were determinedby immunodiffusion according to the method of Johnstone et al. inImmunochemistry in Practice, A. Johnstone and R. Thorpe, eds. BlackwellScientific Publications, Boston, p. 120 (1982).

Results indicated that five of the mAbs tested in the live indirectimmunofluorescence assay were found to give a posteriorly cappedstaining pattern on sporozoites with posteriorly extruded fluorescentmaterial, suggesting the presence of shed antigens. Further, clumpedcollections of fluorescent material free of intact sporozoites were alsoobserved. This indicated that the antigen targets of the five referencedmAbs were being shed in a manner reminiscent of the CSP reactionpreviously reported for malaria (Cochrane et al. J. Immunol. 116:859(1976); Nussenzweig et al. Cell 42:401 (1985)). Additionally, these mAbswere found to bind to oocyst walls. One of the five mAbs, an IgM (kappalight chain) called 3E2, was subsequently examined more closely infunctional assays.

In view of the binding properties of the anti-GP25-200 mAbs disclosedabove, it was of interest to determine whether any of the mAbs alsopossessed the ability to neutralize C. parvum infection. Two differenttypes of neutralizing assays were employed to make this determination.In the first assay, mAbs and sporozoites were combined and incubatedprior to intraintestinal injection into young mice. In the second assay,neonatal mice were orally coadministered with mAb and C. parvum oocysts.Significantly, a positive result in the second assay, wherein the mAbexhibits neutralizing activity, additionally confirms that the mAbretains antigen binding activity under gastrointestinal conditions. Thefollowing Example describes a generalized procedure and presents resultsobtained using the mAb 3E2.

Example 4 describes the first of two methods used for identifying mAbsthat can neutralize C. parvum infection.

EXAMPLE 4 Sporozoite Neutralization Assay

Assays were performed in 96-well round-bottom tissue culture plates(Flow Laboratories). The isolated sporozoite suspension (15 μl) in MEMwas added to each well, mixed with an equal volume of mAb, incubated at37° C. for 30 minutes in a 5% CO₂ atmosphere. Following incubation,sporozoites were suspended by drawing up and then gently expulsing thewell contents 5 times with a 25 μl syringe (Hamilton Co., Reno, Nev.). Atotal of 25 μl from each well containing 2×10⁵ sporozoites was theninjected intraintestinally into 7-day-old specific-pathogen-free BALB/cmice. Uninfected control mice received 25 μl of MEM. Groups of mice wereinjected with sporozoites that had been incubated with isotype controlmAb or 3E2. A 25 μl syringe fitted with a 4 cm length of polyethylenetubing (outer diameter, 0.61 mm; Clay Adams Co., Parsippany, N.J.) wasused for intraintestinal injection. The tubing was inserted 2 cm intothe colon through the anus prior to injection. Following injection, micewere maintained in a 37° C. incubator. Removable rubber patches wereused to prevent defecation for 5 hours postinjection before mice werereturned to their dams. Each group of mice was caged separately incontainers fitted with filter tops.

Mice were euthanized with ether 96 hours postchallenge. The entireintestinal tract was collected immediately after euthanasia, cut into2.5 cm sections, and fixed longitudinally in buffered Formalin.Hematoxylin-eosin stained longitudinal sections representing the entirelength of the intestinal tract from each mouse were prepared, assignedrandomly selected numerical codes, and examined histologically withoutknowledge of treatment, and infection scores were determined. Scores of0, 1, 2, or 3 representing the density of organisms per unit length ofintestinal mucosa were assigned for terminal jejunum and ileum, cecumand colon (0, absence of infection; 1, 1-33% of mucosa parasitized; 2,34-66% of mucosa parasitized; 3, greater than 67% of mucosaparasitized). Cumulative scores ranged from 0 (no infection) to 9(maximum infection) for individual mice. Results of the scoring arepresented in Table 1.

TABLE 1 Neutralization of C. parvum Sporozoite Infectivity by mAb 3E2No. Infected/ Mean Infection Inoculum No. Inoculated Score ± SD P Sp +3E2  0/10 0 <0.0001 Sp + Control IgM 10/10 4.4 ± 1.2

The results in Table 1 indicated that mAb 3E2 had C. parvum neutralizingactivity when the mAb was first combined with sporozoites ex vivo andthen administered intraintestinally. However, a more stringent test ofthe applicability of the neutralizing activity of this mAb preparationinvolved coadministration of oocysts and ascites by the oral route. Thistest is believed to more closely reflect the efficacy of the mAb as itis used as a therapeutic or prophylactic agent. A description of thismore stringent test follows.

Example 5 describes the method used to demonstrate that the mAb 3E2could be administered orally to laboratory animals and retain C. parvumneutralizing activity.

EXAMPLE 5 Neutralizing Activity of mAb 3E2 in vivo

Neonatal BALB/c mice develop intestinal infections following oraladministration of 10⁴ peracetic acid-treated C. parvum oocysts (Riggs etal. Infect. Immun. 62:1927 (1994); Riggs et al. Infect. Immun. 55:2081(1987); Perryman et al. Molec. Biochem. Parasitol. in Press (1996)). Theability of mAb 3E2 to diminish infection was tested by orallyadministering 100 μl of ascites containing the mAb at the time of, aswell as at 2 and every 12 hours thereafter following oral challenge with10⁴ oocysts. Mice were terminated 92 to 94 hours following oocystadministration. Intestinal tracts were removed and scored histologicallyfor the presence and number of C. parvum organisms in epithelial cellsof the removed jejunum and ileum, cecum and colon. Control mice receivedisotype control mAb of irrelevant specificity. Scores of 0-3 wereassigned for each of the three sites, with 0 indicating no organisms; 1,<33% parasitized; 2, 33-66% of cells parasitized; and 3, > than 66% ofcells parasitized. Scores from the three sites were summed to obtain aninfection score for each mouse. Score differences between groups of micewere analyzed by Student's t test. Results of the scoring are presentedin Table 2.

TABLE 2 Protection Against Challenge with C. parvum Oocysts by mAb 3E2Expt. and No. Infected/ Mean Infection Inoculum No. Inoculated Score ±SD P Expt 1 mAb 3E2 4/6 0.7 ± 0.5 <0.0001 Control IgM 8/8 4.5 ± 1.5 Expt2 mAb 3E2 4/7 0.9 ± 0.9 <0.0001 Control IgM 8/8 5.1 ± 1.5

The results in Table 2 demonstrated that orally administeredneutralizing mAb can mediate significant neutralization of C. parvuminfection in vivo.

While mAb 3E2 showed particularly strong neutralizing activity in the invivo assay described above, we discovered that other mAbs specific forC. parvum constituents also could confer significant protection in vivo.The following Example discloses that a collection of mAbs generatedagainst various purified constituents of C. parvum were tested forneutralizing activity in vivo along with one of the samples disclosed inTable 2. The results of the in vivo assay confirmed that neutralizingmAbs can survive gastrointestinal transit and mediate neutralizationduring the short time that zoites are extracellular.

Example 6 describes the methods used to demonstrate that a broadspectrum of C. parvum neutralizing mAbs retained activity in vivo.

EXAMPLE 6 Production and Characterization of a mAb Panel Having C.parvum Neutralizing Activity In Vivo

Native antigens corresponding to CPS-500 and GP-23 were isolated andused according to the methods of Examples 1-3 to prepare and identifyhybridomas secreting anti-C. parvum mAbs. mAb C6B6 was immobilized to asolid matrix to produce an immunoaffinity column that was used to purifyGP-23. Notably, the CPS-500 antigen was non-protein in composition. TheCPS-500 antigen was isolated by silicic acid chromatography according tostandard methods. The mAbs secreted by the hybridomas, together with acollection of mAbs generated against the GP25-200 antigen complex inExamples 1-3, including mAb 3E2, were tested in the in vivo neutralizingassay disclosed in Example 5. Results of the neutralizing assay arepresented in Table 3.

TABLE 3 Immunotherapeutic Effect of Neutralizing mAbs Against IntestinalCryptosporidiosis Mean Infection Score + SD Con- Test trol mAbs PreparedAgainst Isotype mAbs mAbs P 3H2/1B5 GP25-200/GP25-200 —/IgM 2.5 ± 6.3 ±<0.005 2.0 1.3 3E2/4G7 GP25-200/GP25-200 IgM/IgM 1.9 ± 6.3 ± <0.0001 2.01.3 4C11/3A12 GP23/GP25-200 IgM/IgM 3.6 ± 5.7 ± <0.005 0.8 1.5 7D10/3D6GP23/GP25-200 IgG₁/IgM 2.6 ± 5.9 ± <0.001 1.4 1.6 1E10/3E1 GP23/GP25-200IgG₁/IgM 3.6 ± 5.9 ± <0.005 0.5 1.6 3A10/3D10 GP25-200/GP25-200 IgM/IgM3.0 ± 5.7 ± <0.005 0.9 1.5 3B12/3D1 GP25-200/GP23 IgM/IgM 1.1 ± 4.8 ±<0.001 1.6 1.8 7G9/3C8 GP23/GP25-200 IgG_(2a)/IgM 2.4 ± 4.5 ± <0.01 1.61.6 4E11/3B5 GP25-200/GP25-200 IgG₁/IgM 2.1 ± 5.3 ± <0.0001 1.1 1.14H5/7C2 GP25-200/GP23 IgM/IgG_(2a) 0.6 ± 5.2 ± <0.0001 1.0 1.1 3G1/2F5GP25-200/GP25-200 IgM/IgM 5.3 ± 7.0 ± <0.05 1.5 0.7 3E6/4E4GP25-200/GP25-200 IgG₁/IgG₃ 1.8 ± 5.1 ± <0.0005 1.5 1.6 1A9/1F5GP23/GP25-200 IgG₃/IgM 3.8 ± 5.1 ± <0.05 1.4 1.6 7H12 GP23 IgG₁ 5.2 ±6.9 ± <0.005 1.1 0.7 4B10 GP25-200 IgM 3.8 ± 5.2 ± <0.01 1.4 1.1 3E2GP25-200 IgM 0.7 ± 4.5 ± <0.0001 0.5 1.5

The quantitative results presented in Table 3 indicated that several ofthe mAbs raised against purified C. parvum constituents possessedneutralizing activity that was retained following oral administration.These results additionally confirmed that mAb 3E2 possessed superiorneutralizing activity when compared with other mAbs tested in theprocedure described above.

Pools of anti-C. parvum mAbs, including mAb 3E2, optimized forneutralizing activity represent particularly advantageous therapeuticcompositions useful in the practice of the invention. Whereas antigenicdrift could conceivably lead to the loss of an epitope recognized by asingle anti-C. parvum mAb, and corresponding loss of neutralizingactivity of a mAb targeted to that epitope, it is highly unlikely thatantigenic drift would lead to the simultaneous loss of several differentepitopes recognized by a collection of anti-C. parvum mAbs. Accordingly,therapeutic compositions comprising the particularly advantageous mAb3E2 in combination with other mAbs having binding specificities forother C. parvum neutralizing epitopes represent particularly usefulcompositions for providing passive immunity to C. parvum infection.

In view of the particularly advantageous properties of mAb 3E2, anexperiment was conducted to confirm that a composition comprising mAb3E2 in combination with other anti-C. parvum mAbs exhibited strongneutralizing activity in vivo. A pool of three mAbs of irrelevantbinding specificity and having the IgG₁, IgM and IgG₃ isotypes served asa negative control in the procedure. Thus, neutralizing activitydetected in the experimental trial was attributable to the specificityof anti-C. parvum mAbs in the pool. The pool of anti-C. parvum mAbstested in the procedure described below included mAbs 18.44, C6B6, C4A1,2B4, 3D1, 3E2 and M23A1. The results described in the following Exampleclearly indicated that a composition comprising mAb 3E2 and otheranti-C. parvum mAbs exhibited strong neutralizing activity in vivo.

Example 7 describes the method used to demonstrate that a cocktail ofanti-C. parvum mAbs exhibited strong neutralizing activity in vivo.

EXAMPLE 7 Additive Therapeutic Effect of mAb Pools Targeting MultipleNeutralization-Sensitive C. parvum Epitopes

Hybridomas secreting mAbs listed in the first column of Table 4 wereseparately propagated in BALB/c mice and ascites fluid samplescontaining the mAbs collected using standard laboratory methods. ThesemAb preparations were administered to neonatal BALB/c mice as pools ofascites fluid to test the in vivo neutralizing capacity of thepreparations according to the method described under Example 5. Thevolume of ascites fluid administered to neonatal mice in this procedurewas held constant at 150 μl. Equal volumes of different ascites fluidsamples were mixed to create preparations of mAb combinations. A pool orcollection of mAbs HL113 (IgM), HL245 (IgG₃) and HL296 (IgG₁), allhaving irrelevant binding specificities, served as a negative control inthe procedure. A second pool included mAbs 18.44, C6B6, C4A1, 2B4, 3D1,3E2 and M23A1, all having binding specificities for epitopes disposed onC. parvum. In this procedure, 8-10 mice were used for each test group.

The results of the procedure testing the neutralizing activity of pooledmAbs are presented in Table 4. Plus and minus signs in the Tableindicate the antigens against which mAbs in the pools were generated.The negative control mAbs HL113, HL245 and HL296 were not preparedagainst any of CPS-500, GP-23 or GP25-200, and do not bind to C. parvum.The mAbs 18.44 and 2B4 had binding specificities directed againstCPS-500; mAbs C6B6 and 3D1 had binding specificities directed againstGP-23; while mAbs C4A1 and 3E2 had binding specificities directedagainst GP-25-200. The mAb M23A1 was prepared against the merozoitestage (MZ in the Table).

TABLE 4 Therapeutic Effect of mAb Pools Targeting MultipleNeutralization-Sensitive C. Parvum Epitopes Mean PREPARED InfectionAGAINST Score ± mAb Pool CPS-500 GP-23 GP25-200 MZ^(c) SD HL113/HL245/ −− − − 4.8 ± 0.4 HL296 (isotype controls) 18.44/2B4 +/+ C6B6/3D1 +/+C4A1/3E2 +/+ M23A1 + 1.4 ± 1.0^(a,b) ^(a)P < 0.0001 compared to control^(b)P < 0.0001 compared to 18.44/C6B6/C4A1 (3 mAbs originally used todefine CPS-500, P23 and GP25-200, respectively) ^(c)merozoite stage

The results presented in Table 4 demonstrated that a pool or collectionof anti-C. parvum mAbs that included mAb 3E2 was useful as a therapeuticcomposition having C. parvum neutralizing activity. More specifically,the mean infection score of 4.8±0.4 obtained in the negative controltrial represented a baseline for infectious activity. The mean infectionscore was dramatically reduced to a value of 1.4±1.0 in mice receiving acomposition comprising mAb 3E2 in combination with other C. parvumneutralizing mAbs. Thus, in this experiment the mean infection scoremeasured in animals receiving a pool of mAbs that included mAb 3E2 wasapproximately 30% of the score measured in animals receiving poolednegative control mAbs. While this level of reduction was significant, itwas not as dramatically reduced as it was in animals that received mAb3E2 in the experiment presented in Table 2 and 3. More particularly, thegroup of animals that received mAb 3E2 alone showed mean infectionscores representing only about 15% of the negative control value. Inaggregate, these results indicated that mAb 3E2 alone exhibitedunexpectedly strong C. parvum neutralizing activity and thatcompositions including mAb 3E2 in combination with other mAbs directedto different neutralizing epitopes also exhibited strong neutralizingactivity.

Example 8 describes the method used to demonstrate that mAb 3E2 hadbinding specificity for a carbohydrate/carbohydrate-dependent structure.

EXAMPLE 8 The Antigenic Target of mAb 3E2 Comprises a Carbohydrate

The method described by Woodward et al. in J. Immunol. Methods 78:143(1985) was modified by using 2-12% SDS-PAGE as well as 10-20% SDS-PAGEseparations to examine the chemical identity of the epitope recognizedby mAb 3E2. More specifically, Western blotted protein samples fromsolubilized sporozoites were either left as untreated controls ortreated with 5 mM periodate prior to detection with the mAb 3E2.

Results of these procedures indicated that mAb 3E2 had bindingspecificity for a carbohydrate/carbohydrate-dependent moiety disposed ona glycoprotein. The untreated control lanes of the blot that had beenprobed with 3E2 revealed multiple bands having molecular weights ofapproximately 46, 51, 70, 84, 106,112, 117,120, 134, 147, 154, 158, 172,183, 220, 230, 265, and a diffuse band in the size range of 1200-1400kDa. However, no staining pattern was detected in the sample lane thatreceived periodate treatment. This indicated that the epitope recognizedby the mAb probe was destroyed by periodate oxidation and had acarbohydrate/carbohydrate dependent structure. The observations that thetarget of mAb 3E2 binding was periodate sensitive; could be visualizedon Western blots and could be stained by protein-detecting reagents(Example 1) all indicated that 3E2 had binding specificity for acarbohydrate/carbohydrate-dependent structure disposed on a glycoproteinmolecule.

As indicated above, the neutralizing mAb 3E2 will find importantapplication in the control and prevention of cryptosporidiosis inhumans, including patients infected with HIV (AIDS). Routes ofadministration for pharmaceutical compositions that include mAb 3E2include oral and intraintestinal administration according to methodsthat will be apparent to those having ordinary skill in the art.Alternative methods of delivery of the therapeutic mAb 3E2 preparationinclude: lyophilized preparations; alternative carrier systems includingdispersion into colostrum; encapsulation; and time-release encapsulationfor oral administration and post gastric release. Effective dosages maybe empirically determined, but will ordinarily be sufficiently high thatoocyst levels detectable in fecal samples will decrease substantiallywithin a few days following the first administration of the mAb 3E2containing composition. Finally, there are no known adverse side effectsknown to accompany administration of the mAb 3E2 composition and it isbelieved that individuals may be administered with the composition untildiarrhea symptoms dissipate or indefinitely.

Example 9 describes how a preparation including the mAb 3E2 can beadministered to a human patient infected by C. parvum.

EXAMPLE 9 Method of Providing Passive Immunity to Infection by C. parvum

An AIDS patient experiencing diarrhea as the result of infection with C.parvum is first identified. A baseline measurement indicates thepresence of high numbers of oocysts present in a fecal sample.

The patient is administered with a buffered preparation containingbovine colostrum and mAb 3E2 in an amount of up to 1 g of the mAb. ThemAb has been produced in a bioreactor under serum-free conditions and isnot associated with infectious agents. The colostrum serves as a carrierfor the mAb and provides a source of stabilizing agents that preservethe neutralizing activity of the mAb. The patient is administered withthe preparation twice daily.

The patient's clinical parameters show improvement within a few daysfollowing administration of the mAb-containing preparation. Thepatient's diarrhea dissipates and the oocyst quantitation in a fecalsample is decreased to a very low level. These results confirm that oraladministration of a preparation including the mAb 3E2 is of therapeuticvalue.

Although the foregoing Example provides a description of a therapeuticapplication of the mAb 3E2, prophylactic applications wherein anindividual receives the mAb prior to C. parvum exposure can also becarried out.

Experimental results presented above indicated that mAb 3E2 was one offive mAbs directed against the GP25-200 and high molecular weightglycoprotein that stimulated the CSP-like reaction in sporozoites whentested in a live indirect immunofluorescence assay. This CSP-likereaction was characterized by progressive extrusion and eventualshedding of membranous material from the sporozoite posterior andresembled the CSP reaction described for malarial sporozoites byCochrane et al. in J. Immunol. 116:859 (1976). Progressive rounding ofC. parvum sporozoites after contacting mAb 3E2 suggested osmoregulatorydysfunction or perhaps premature triggering to the trophozoite stage.The CSP-like reaction was initiated at the apical end of C. parvumsporozoites and posterior translocation of the antigen was inhibitableby treatment of sporozoites with 5 μM cytochalasin-D. This latterobservation indicated that the reaction was dependent on an intactcytoskeletal apparatus and microfilaments.

Significantly, we have also determined that the merozoite stage of C.parvum undergoes the CSP-like reaction upon contacting mAb 3E2. TheCSP-like reaction observed in the merozoite was morphologicallyindistinguishable from that which occurred in the sporozoite stage. AllmAbs which elicited the CSP-like reaction did so within seconds ofcontacting sporozoites or merozoites in vitro and provided near completeprotection against infectivity in vivo.

Interestingly, the epitope recognized by mAb 3E2 was localized tosporozoite apical complex organelles, including electron-dense granules.More particularly, immunoelectron microscopy performed using either mAb3E2 or an isotype-matched control IgM mAb and sporozoites showed thatelectron-dense granules were predominantly labeled by mAb 3E2.

Example 10 describes the method used to localize the antigen bearing theepitope recognized by mAb 3E2 prior to and during the CSP-likereactions.

EXAMPLE 10 Localization of the Epitope Recognized by mAb 3E2

Sporozoites were purified by anion exchange chromatography according tothe method described by Riggs et al. in Infect. Immun. 55:2081 (1987),and either fixed immediately for immunoelectron microscopy in a solutionof 2% [v/v] paraformaldehyde and 0.5% [v/v] glutaraldehyde in HBSS for aperiod of 15 minutes, or first incubated with mAb 3E2 or isotype-matchedcontrol mAb, allowed to undergo the CSP-like reaction and then washedand fixed. Samples were washed twice in HBSS, dehydrated with 95%ethanol and embedded in LR white resin. Sections were mounted on nickelgrids and blocked with PBS made with 0.1% (w/v) BSA and 0.1% (v/v) TWEEN20 for 10 minutes at 21° C. Sporozoites that had been fixed and embeddedwithout exposure to mAb 3E2 while viable were incubated overnight at 4°C. on drops of mAb 3E2 or isotype-matched control, washed four timeswith PBS, and incubated with affinity-purified rabbit anti-mouse IgM(Zymed, San Francisco, Calif.), washed again, and incubated withaffinity-purified colloidal gold conjugated goat anti-rabbit IgG (Zymed,20 nm or 5 nm, EM grade). Finally, grids were washed and post-fixed withan aqueous solution of 4% (w/v) formaldehyde, 1% (v/v) glutaraldehydefor studies localizing the antigenic target of mAb 3E2. Sporozoiteswhich had been exposed to 3E2 prior to fixation and embedding wereblocked and incubated with rabbit anti-mouse IgM and colloidal goldantibodies as above to provide a means of visualizing the CSP-likereaction. Immunostained samples were observed and photographed with aJEOL 100 CX EM at 80 KV.

Results indicated dense mAb 3E2 immunogold labelling of apicalorganelles, including electron-dense granules. Identification oflabelled organelles as apical complex, including dense granules wasconfirmed by comparison with the published ultrastructural morphologypresented by Current et al. in J. Protozool 33:98 (1986) and Fayer etal. in J P Dubey, C A Speer and R Fayer (eds.), Cryptosporidiosis of Manand Animals, CRC Press, Ch 1 (1990).

In view of the desirable properties of mAb 3E2 and the other four mAbsthat similarly stimulated the CSP-like reaction in C. parvum, it was ofinterest to better assess the properties that were shared by these mAbs.Although not explicitly shown below, our finding that mAbs 3E2, 3B12,3A12, 3A11 and 3E6 all gave identical patterns on Western blots and byindirect immunofluorescence suggested that the mAbs recognized the sameantigen.

As disclosed in the following Example, all five mAbs having the abilityto stimulate the CSP-like reaction bound an epitope that was identicalto, or closely related to, the epitope bound by mAb 3E2. Morespecifically, the results disclosed in the following two Examplesdemonstrated that mAbs which stimulated the CSP-like reaction alsocompeted with mAb 3E2 for antigen binding. Accordingly, we discoveredthat the ability to compete with mAb 3E2 for binding to the GP25-200 and1,200-1,400 KDa antigen target represented a useful criterion foridentifying mAbs having the ability to stimulate the CSP-like reaction.

Example 11 describes one method used to demonstrate that mAbs having theability to stimulate the CSP-like reaction also had bindingspecificities similar to that of mAb 3E2.

EXAMPLE 11 Competition ELISA Using Biotinylated mAb 3E2 andPeroxidase-Labelled Streptavidin for Detection

Purified sporozoites dispersed in PBS containing protease inhibitorswere disrupted by sonication and repetitive freeze-thaw cycles accordingto standard methods. The lysate was centrifuged at 16,000×g for 30minutes (4° C.), and the supernatant isolated. The individual wells of96 well plates were then coated with the equivalent of 5×10⁵ solubilizedsporozoites/well for a period of 12 hours (4° C.). The wells werewashed, blocked with fish gelatin (3.5% w/v) and washed again, allaccording to standard laboratory methods. Wells in triplicate were thenincubated for 1 hour either with unlabeled, individual: (1) mAbs 3E2,3B12, 3E6, 3A12 or 3A11; (2) mAbs 1G2, 2D10, 7B6, C6B6 or C4A1, all ofwhich recognized C. parvum epitopes distinct from the epitopesrecognized by mAbs in the preceding group based on results obtained fromWestern blotting, and none of which elicited the CSP-like reaction; or(3) IgM isotope matched control mAb HL113 having an irrelevant bindingspecificity. All mAb reagents were added in volumes of 100 μl/well to afinal concentration of 30 μg/ml of mAb. Following incubation for 1 hour,biotinylated mAb 3E2 was added in a volume of 25 μl/well to give a finalmAb 3E2 concentration of 1 μg/ml. Samples were incubated an additional30 minutes, washed, incubated 30 minutes with peroxidase-labelledstreptavidin (100 μl/well of a 0.5 μg/ml solution), washed and exposedto ABTS peroxidase substrate according to the method described by themanufacturer (Kirkegaard and Perry). Absorbances at a 405 nm wavelength(OD₄₀₅) were read on an ELISA plate reader and are presented in Table 5.Notably, each data point presented in Table 5 represents the mean offive replicate wells.

TABLE 5 Competition ELISA Using Biotinylated mAb 3E2 andPeroxidase-Labeled Streptavidin for Detection mAb Type Unlabeled mAbOD₄₀₅ Control HL113 0.91 IG2 0.85 7B6 0.78 C6B6 0.84 C4A1 0.83CSP-Stimulatory 3E2 0.10 3B12 0.15 3E6 0.09 3A12 0.15 3A11 0.10

The results presented in Table 5 indicated that all of the mAbs havingthe ability to stimulate the CSP-like reaction recognized the same,similar or overlapping epitopes as that recognized by mAb 3E2. Moreparticularly, mAbs 3E2, 3B12, 3E6, 3A12 and 3A11 all reduced the amountof biotinylated mAb 3E2 that was immobilized in the wells of the ELISAplate as reflected by less significant production of the coloredreaction product detectable at the 405 nm wavelength. Significantly,none of the control mAbs recognized the same epitope as that recognizedby mAb 3E2 based on Western blotting results. Moreover, control mAbsHL113, IG2, 7B6, C6B6 and C4A1 showed no evidence for competition withmAb 3E2. The entry corresponding to the unlabeled mAb 3E2 in Table 5represented a perfect competitor for the biotinylated mAb 3E2 while thenegative control mAb HL113 represented no competition.

Example 12 describes a second method useful for demonstrating that mAbscapable of eliciting the CSP-like reaction in C. parvum also recognizedthe same, similar or overlapping epitope as that recognized by mAb 3E2.The results of this radioimmuno assay (RIA) procedure confirmed thefindings presented in the preceding Example.

EXAMPLE 12 Competition RIA using ¹²⁵I-Labelled mAb 3E2 for Detection

Immulon-4 removable wells were coated with the equivalent of 5×10⁵purified sporozoites/well exactly as described in the preceding Example.Wells, in replicates of five, were then washed, blocked with PBS-BSA (2%w/v), washed again, incubated for 3 hours (37° C.) with unlabeled,individual: (1) mAbs 3E2, 3B12, 3A12 or 3A11; or (2) mAbs C6B6, 7D10,4D5, 1D8, 3A3, 1B5, IG1, 3A7 or 3H1. The mAbs in the second group allrecognized C. parvum epitopes distinct from the epitopes recognized bythe mAbs in the first group based on the results of Western blotting.None of the mAbs in the second group elicited the CSP-like reaction. AllmAbs were added in a volume of 200 μl/well with a final concentration of8 μg/ml of the mAb. Samples were incubated for 1 hour (37° C.) with¹²⁵I-labeled (Iodogen, Pierce) mAb 3E2 (50 μl/well, 2 μg/ml finalconcentration of mAb), washed, and counted in a gamma counter. Resultsrepresenting the mean of five replicate wells are presented in Table 6.

TABLE 6 Competition RIA Using Radiolabeled mAb 3E2 for DetectionImmobilized Radioactivity mAb Type Unlabeled mAb (CPM) Control C6B614,599 7D10 12,655 4D5 14,664 1D8 14,340 3A3 13,961 1B5 13,257 IG215,581 3A7 13,341 3H1 15,061 CSP-Stimulatory 3E2  1,364 3B12  1,870 3A12 3,845 3A11  2,451

The results presented in Table 6 again indicated that mAbs whichstimulated the CSP-like reaction also competed with mAb 3E2 for antigenbinding. None of the unlabeled control mAbs in Table 6 showed evidencefor competition with radiolabeled mAb 3E2 for epitope binding. All ofthe samples that included control mAbs gave levels of immobilized,radiolabeled mAb 3E2 substantially higher than the level observed whenunlabeled mAb 3E2 was used as the competitor. Thus, mAb 3E2 served as apositive control for the competition while the control mAbs representednon-competing negative controls in the procedure. All of the mAbs havingCSP-like reaction stimulatory activity led to reductions in the level ofimmobilized, radiolabeled mAb 3E2, thereby providing evidence forbinding competition with radiolabeled mAb 3E2. These inhibition resultsindicated that mAbs 3E2, 3B12, 3A12 and 3A11 recognized the same,similar, or overlapping epitopes.

Thus, the results of the competition binding assays presented in the twopreceding Examples confirmed an absolute correlation between mAbs thatcompeted with mAb 3E2 for antigen binding and mAbs that stimulated theCSP-like reaction. It follows that any mAb that has the ability tocompete with mAb 3E2 for epitope-binding will also stimulate theCSP-like reaction. In view of these findings and conclusions, wereasoned that the antigenic target of mAb 3E2 played a critical role inthe mechanism of the CSP-like reaction. Accordingly, we proceeded toidentify and isolate the molecular species bound by mAb 3E2.

Example 13 describes the method used to demonstrate that membranousmaterial shed by sporozoites after contacting mAb 3E2 was of highmolecular weight and was highly glycosylated. Glycosylation status ofthe shed material was inferred from: (a) the carbohydrate nature of thetarget epitope, (b) highly variable migration of the material duringSDS-PAGE typical of glycoproteins, and (c) a significant decrease inmolecular weight following deglycosylation and separation by SDS-PAGE.Also disclosed is the discovery that the high molecular weight speciesmechanistically involved in the CSP-like reaction is an exoantigenconstitutively released by infectious sporozoites.

EXAMPLE 13 PERCOLL Density Gradient Purification of Membranous MaterialShed After the mAb 3E2-Induced CSP-Like Reaction

mAb 3E2 (288 μg) was incubated with 5×10⁸ excysted oocysts for a periodof 1 hour (37° C.) until the CSP-like reaction, characterized bymembranous precipitate formation on sporozoites, was maximal. A 1 mlsample of the preparation was layered onto 9.0 mls of a PERCOLL(Pharmacia, Piscataway, N.J.) solution consisting of 9 parts PERCOLL, 1part 10× Alsever's solution (4.2 g NaCl/liter; 8.0 g trisodium citratedihydrate/liter; 20.5 g glucose/liter) purchased from GIBCO-BRL (GrandIsland, N.Y.), and 9 parts 1× Alsever's solution in a centrifuge tube.Membranous precipitates were recovered from the gradient followingcentrifugation at 40,000×g for 30 minutes (4° C.). Gradient fractionswere removed sequentially and examined by phase contrast microscopy forthe presence of membranous precipitates. The preparation separated intofive zones. In order from lowest to highest density, these zonesrepresented: (1) oocyst walls, (2) membranous precipitates, (3)precipitates attached to sporozoites, (4) sporozoites and (5) intactoocysts. Precipitate-containing fractions were washed four times bycentrifugation at 16,000×g for 10 minutes (4° C.) using PBS containingprotease inhibitors (5.0 mM EDTA, 5.0 mM iodoacetamide, 0.1 mMN-tosyl-L-lysl chloromethyl ketone, 1.0 mM phenylmethylsulfonylfluoride) and then subjected to 6-8 sequential PERCOLL gradients untilthe precipitate fraction was relatively free of sporozoite, intactoocyst, and oocyst wall contamination. Purified membranous precipitateswere pelleted at 16,000×g for 10 minutes (4° C.), resuspended in 0.03 mllysis buffer (50 mM Tris, 5.0 mM EDTA, 5.0 mM iodoacetamide, 0.1 mMN-tosyl-L-lysl chloromethyl ketone, 1.0 mM phenylmethyl-sulfonylfluoride, and 1% [wt/vol] n-octyl-B-D-glucopyranoside), solubilized for30 minutes (4° C.) and stored at −80° C. until analysis.

A silver stained 2-12% gradient SDS-PAGE reducing gel demonstrated thepresence of a single prominent, approximately 1400 kDa band (CSL) inmembranous material shed from viable sporozoites during the CSP-likereaction. Lower molecular weight bands co-migrated with IgM mAbcontrols, indicating that CSL was the antigen species recognized by mAb3E2 during the CSP-like reaction.

The results presented in the foregoing Example indicated that themechanism of the mAb 3E2 stimulated CSP-like reaction was associatedwith the shedding of proteinaceous material in the molecular weight sizerange of from 1200-1700 kDa when measured by SDS polyacrylamide gelelectrophoresis. As indicated by the findings presented in the followingExample, we discovered that the shed proteinaceous material contained anepitope that was specifically bound by mAb 3E2. Thus, the antigenictarget of a mAb having the ability to induce the CSP-like reaction insporozoites of C. parvum was localized to high molecular weight materialshed during the CSP-like reaction.

Example 14 describes the methods used to demonstrate that the epitoperecognized by mAb 3E2 was expressed on different developmental stages ofC. parvum and that this material was shed during the CSP-like reaction.The molecular species bound by mAb 3E2 during the CSP-like reaction,from among the multiple bands recognized by mAb 3E2 in whole sporozoitesis referred to below as the CSL protein.

EXAMPLE 14 The CSL Protein Shed from Sporozoites After the CSP-LikeReaction is also Present in Excysted Oocysts and Whole Sporozoites

Membranous material released from approximately 9×10⁷ sporozoites afterthe mAb 3E2-induced CSP-like reaction was isolated by PERCOLL densitygradient centrifugation as described in the preceding Example. Thematerial was washed and electrophoretically separated on a 2-12% SDSpolyacrylamide gel under reducing conditions. Bioreactor-derived (serumfree), purified mAb 3E2 (15 μg), purified sporozoites (3-7.5×10⁶),sporozoite antigen purified by mAb C4A1 affinity chromatography (2 μgpurified protein), and the soluble phase of a viable sporozoite culturesupernatant (2 μg protein) harvested by ultracentrifugation (100,000×g,1 hour) and filtration (0.2 μm pore size) after two hours of incubationin the absence of mAb, were electrophoresed and silver stained forcomparison. All preparations were solubilized in lysis buffer containingprotease inhibitors prior to electrophoresis. Molecular weight standardsused were titin (2,450 kDa); nebulin (770 kDa); and myosin (208 kDa).

Results indicated that a prominent C. parvum derived band having amolecular weight of approximately 1,400 kDa was present in purifiedmembranous precipitates shed during the CSP-like reaction. This band,which represented the CSL high molecular weight exoantigen, co-migratedwith a band of the same molecular weight derived from (1) purified wholesporozoites, (2) mAb C4A1 affinity chromatography purified sporozoiteantigen (containing a subset of whole sporozoite bands), and (3) thesoluble phase of viable sporozoite culture supernatant. Several otherlower molecular weight bands present in the membranous precipitatepreparation co-migrated with bands of similar molecular weight derivedfrom purified mAb 3E2, indicating that they were of nonsporozoite origin(mAb heavy and light chains). The results also indicated that the highmolecular weight CSL antigen which harbored the mAb 3E2 binding site wasthe major band in both PERCOLL-purified precipitates and soluble phaseof sporozoite culture supernatant. These findings indicated that theantigenic target of mAb 3E2 specifically involved in the CSP-likereaction had been identified and purified and was constitutivelyreleased by sporozoites in culture.

As described in the following Example, Western blotting demonstratedthat a single species having a molecular weight of approximately 1400kDa and immunoreactive with mAb 3E2 was present in PERCOLLgradient-isolated membranous precipitate shed from viable sporozoitesafter the CSP-like reaction. The high molecular weight species was theonly reactive band in precipitates derived from sporozoites. Thisspecies corresponded in size to the 1400 kDa protein band observed insilver-stained gels and was immunoreactive with both mAbs 3E2 and C4A1.This band co-migrated with an immunoreactive band of the same molecularweight that was present among a collection of immunoreactive bands insolubilized whole sporozoite preparations.

Example 15 describes the methods used to demonstrate that mAb 3E2 bindsthe CSL glycoprotein antigen directly.

EXAMPLE 15 The CSL Glycoprotein Harbors the Epitope Recognized by mAb3E2

Solubilized sporozoites or shed precipitates were resolved by reducingSDS-PAGE in a 2-12% gradient gel and blotted onto a nitrocellulosemembrane. A control lane was loaded with 25 μg mAb 3E2 alone. Lanes wereprobed with mAb 3E2, mAb C4A1 or an isotype control mAb.Affinity-purified, alkaline phosphatase-conjugated goat anti-mouse IgMantibody was used to detect binding following addition of substrate.

Results indicated that the only immunoreactive bands other than the 1400kDa band in lanes containing purified membranous precipitatesco-migrated with mouse mAb IgM bands, and therefore represented mAb 3E2components in shed material. Immunoreactive, parasite-derived lowmolecular weight bands in PERCOLL-purified precipitates, which may nothave been visualized in Western blots of 2-12% gels, were not detectedin Western blots of purified precipitates resolved in 10-20% gels. Theresults indicated that the approximately 1400 kDa band was the onlyantigen bound by mAb 3E2 during the CSP-like reaction, and thereforemust be mechanistically involved in the reaction. mAb C4A1, which didnot elicit the CSP-like reaction, recognized an epitope on theapproximately 1400 kDa antigen different from the epitope recognized bymAb 3E2.

As described in the following Example, a two-site immunoradiometricassay (IRMA) was employed to confirm that the epitope disposed on thehigh molecular weight target antigen of mAb 3E2 was repetitive. Thisfeature of the epitope recognized by mAb 3E2 advantageously facilitatedimmunoprecipitation of the target antigen by the mAb in the absence of asecondary antibody.

Example 16 describes the method used to confirm that the epitoperecognized by mAb 3E2, and disposed on the high molecular weight CSLantigen, was a repetitive epitope.

EXAMPLE 16 The Epitope Disposed on the CSL Glycoprotein and Recognizedby mAb 3E2 is Repetitive

Two-site IRMA was carried out according to the method described byCochrane et al. in Infect. Immun. 45:592 (1984), with the followingmodifications. Removable Immulon 4 wells in microtiter plate frames(Dynatech, Chantilly, Va.) were coated for 12 hours (4° C.) withpurified mAb 3E2 or purified IgM control mAb of irrelevant specificitydiluted in PBS (25 μg mAb/ml final concentration), washed, blocked (2%[w/v] BSA in PBS), and washed again, all according to standardlaboratory methods. Constitutively released CSL high molecular weightexoantigen was obtained for analysis by incubating purified viablesporozoites in Hank's buffered saline solution (HBSS) for 2 hours (37°C.). The preparation was then cooled to 4° C. and centrifuged at 5000×gfor 10 minutes (4° C.) to remove sporozoites. The exoantigen-containingsupernatant was ultracentrifuged at 100,000×g for 20 minutes (4° C.) andpassed through a syringe-tip filter having a 0.2 μm pore size to removeresidual insoluble material. Following the addition of proteaseinhibitors phenylmethylsulfonyl fluoride, N-tosyl-L-lysl chloromethylketone, and iodoacetamide, samples were stored frozen at −80° C. untiluse.

The CSL high molecular weight soluble exoantigen released from 8×10⁶viable sporozoites was added to each of ten replicate mAb 3E2- orcontrol IgM mAb-coated wells in 200 μl PBS and incubated 2 hours (37°C.). Free antigen was removed by washing, and wells were then incubatedwith ¹²⁵I-labeled mab 3E2 (2×10⁵ TCA precipitable cpm/well) for 1 hourat 37° C. After washing, ¹²⁵I-labeled mAb 3E2 specifically bound to CSLhigh molecular weight exoantigen which had been immobilized to the mAb3E2-coated wells. Immobilized radioactivity was quantitated as “countsper minute” (cpm) using a gamma counter. Results indicated that54,317±2148 cpm (n=10) of radiolabeled mAb 3E2 was immobilized for mAb3E2 coated wells but only 2823±186 cpm (n=10) was immobilized for wellscoated with the IgM control mAb. These results indicated that theepitope recognized by mAb 3E2 occurred at least twice on the CSL highmolecular weight exoantigen that was mechanistically involved in theCSP-like reaction.

Constitutive release of the CSL high molecular weight exoantigen fromsporozoites was demonstrated by immunoprecipitation of theradioiodinated soluble phase of viable sporozoite culture supernatant bymAb 3E2-Sepharose, SDS-PAGE, and autoradiography. Results from theseprocedures indicated that mAb 3E2 specifically precipitated a single,prominent, approximately 1400 kDa radioiodinated antigen from thesoluble phase of cultured sporozoites as revealed by resolution ofimmunoprecipitated material in reducing 2-12% SDS-PAGE followed byautoradiography. Control mAb IgM-Sepharose of irrelevant specificity didnot precipitate this antigen. Further, silver stained SDS-PAGE reducinggels of the soluble phase of sporozoite culture supernatant contained amajor, approximately 1400 kDa band, substantially free of otherproteins.

Example 17 describes a method useful for purifying the high molecularweight CSL antigen that was involved in the CSP-like reaction and thatwas recognized by mAb 3E2.

EXAMPLE 17 Isoelectric Focussing Method of Purifying the High MolecularWeight Antigen Mechanistically Involved in the CSP-Like Reaction

Excysted oocysts were solubilized by freeze-thaw and sonication in thepresence of protease inhibitors and 1% w/v octyl glucoside. The solublefraction was collected by centrifugation at 5000×g for 10 minutes (4°C.) and dialyzed for 12 hours against distilled water using a12,000-14,000 molecular weight cut-off membrane. A Rotofor (Bio-Rad)isoelectric focusing chamber was then used to isolate the high molecularweight exoantigen from the solubilized, dialyzed sporozoite preparation.The Rotofor apparatus was prepared by addition of 0.1 M phosphoric acid(pH 1.2) to the cationic chamber, addition of 0.1 M NaOH (pH 12.0) tothe anionic chamber, and addition of HPLC-quality water containing 3%ampholines (pH 3.5-5.0 range) to the focussing chamber. The apparatuswas maintained at 4° C. and connected to a 12-watt power source for 1hour to establish a pH gradient. The solubilized, dialyzed sporozoitepreparation (2 ml) was then added and isoelectric focussing allowed toproceed for 6 hour at 12 watts. At the completion of the focussingcycle, 20 fractions spanning the separated protein range were collected,neutralized, and analyzed for presence and purity of the high molecularweight antigen by Western blotting and SDS-PAGE/silver staining.Refocusing of fractions containing the high molecular weight antigenwere performed until immunoreactive, pure antigen was obtained.

The results of these procedures demonstrated that the exoantigen targetof mAb 3E2 could be isolated from all other sporozoite antigen species,including those recognized by mAb 3E2, and that the purificationprocedures employed in this process did not destroy immunoreactivity ofthe antigen. More particularly, a silver stained 2-12% gradient SDS-PAGEreducing gel of solubilized C. parvum before isoelectric focussingrevealed the presence of greater than 50 bands in the 7-2,450 kDa range,including the CSL high molecular weight exoantigen. After isoelectricfocusing, a single band having a molecular weight of approximately 1,400kDa was visualized by silver staining 2-12% SDS-PAGE gels of purifiedantigen. Western blotting of the purified sample demonstrated a single,approximately 1,400 kDa band immunoreactive with mAb 3E2. The purifiedantigen co-migrated with an antigen of the same molecular weight fromamong the multiple antigens recognized by mAb 3E2 in whole C. parvumpreparations.

As described in the following Example, purified CSL glycoproteinreactive with mAb 3E2 and isolated from oocysts by preparative SDS-PAGEwas shown to function as an immunogen capable of stimulating an anti-C.parvum response in an animal. While the Example describes a particularprotocol, alternative immunization protocols also are expected to beuseful for stimulating an anti-C. parvum immune response in an animal.For example, we contemplate that adjuvants familiar to those havingordinary skill in the art can be administered in combination withantigen to stimulate vigorous anti-C. parvum immune responses. Aparticular protocol useful for immunizing cows and other ruminants canbe found in Infect. Immun. 62:1927 (1994). According to this protocolantigen is combined with Ribi TDM-CWS adjuvant (Ribi ImmunochemResearch, Hamilton, Mont.) and administered subcutaneously (in theshoulder) and intramuscularly (in the hip) at timed intervals. Thisprotocol is particularly useful for stimulating the production ofhyperimmune colostrum useful for conferring passive immunity in calvesand humans when the colostrum is administered orally. The carrier usedfor dispersing the purified antigen of the invention can be a bufferedsaline solution, such as phosphate buffered saline, and optionally caninclude an adjuvant such as Freund's adjuvant or the Ribi adjuvant.Other commercially available adjuvants also are contemplated for usewith the invention to stimulate a strong anti-C. parvum immune response.If the purified antigen is a weak immunogen in a particular application,then the antigen can be coupled to a carrier protein such as keyholelimpet hemocyanin to improve the immunogenicity of the purified CSLglycoprotein shown herein to be immunoreactive with mAb 3E2.

Example 18 describes the method that was used to stimulate activeimmunity in an animal. While the Example makes explicit reference toimmunization of a chicken, the method is equally adaptable toimmunization of other animals, including mammals.

EXAMPLE 18 Method of Stimulating an Anti-C. parvum Immune Response in anAnimal

CSL glycoprotein was purified from oocysts by preparative SDSpolyacrylamide gel electrophoresis. SDS-PAGE and silver staining wasused to confirm purity of the CSL glycoprotein sample. Western blotanalysis confirmed that reactivity of CSL with mAb 3E2 was preservedfollowing the purification procedure. Chickens were hyperimmunized withthe purified CSL glycoprotein according to standard methods. Moreparticularly, the purified CSL glycoprotein antigen was combined withFreund's complete adjuvant and administered subcutaneously andintramuscularly for the first immunization at approximately two monthsof age. The combination of antigen and Freund's incomplete adjuvant wasadministered subcutaneously and intramuscularly at four week intervalsfor between 5 and 7 rounds of immunization. Each immunization consistedof 1 ml total volume and was split equally between the subcutaneous andintramuscular injections. Serum antibody responses in the hyperimmunizedchickens were monitored by indirect immunofluorescence and Westernblotting, as described above.

Results of these procedures indicated that chickens hyperimmunized withthe purified CSL glycoprotein advantageously exhibited high titers ofserum antibodies directed against sporozoites. Western blottingprocedures indicated that the anti-CSL antibodies were present in serumsamples isolated from the hyperimmunized chickens. Interestingly, thechickens also developed antibody responses against multiple, lowermolecular weight sporozoite antigens. This latter finding indicated thepresence of cross-reactive epitopes on the CSL glycoprotein used as animmunogen and the lower molecular weight species. Importantly, theanti-CSL sera from the hyperimmunized chickens was also shown to inducethe CSP-like reaction in live sporozoites.

These results clearly indicated that an immunogenic composition whichincluded a substantially purified C. parvum antigen specificallyrecognizable by mAb 3E2 and a pharmaceutically acceptable carrier hadutility in a method for stimulating an anti-C. parvum immune response inan animal. The method of stimulating an anti-C. parvum immune responseinvolved obtaining the immunogenic composition and administering theimmunogenic composition to the animal according to a vaccinationprotocol. Pharmaceutically acceptable carriers such as saline solutions,optionally including one or more adjuvants, could be co-injected withthe CSL antigen to aid in delivery of the immunogen and in stimulating avigorous immune response.

Example 19 describes one method of stimulating an active immune responseagainst C. parvum based on immunization with the CSL constituent of theGP25-200 complex. Although this Example describes the vaccination of ahuman subject, the method can also be adapted for use in immunizinganimals, such as cattle or other livestock. Those having ordinary skillin the art will recognize that other methods for performing avaccination are well known and can be used in connection with thepurified high molecular weight CSL antigen constituent of the GP25-200complex.

EXAMPLE 19 Vaccine Composition Comprising the CSL Glycoprotein

A human subject at risk of exposure to C. parvum is first identified.The subject is injected subcutaneously with an immunogenic compositioncomprising purified CSL high molecular weight antigen reactive with mAb3E2, wherein the antigen is produced in accordance with the method ofExamples 17 and 18. During preparation of the immunogenic composition, asample containing the purified CSL high molecular weight antigen isfirst dialyzed against physiological saline, filter-sterilized,concentrated and combined with a pharmaceutically acceptable carrier. Anadjuvant may optionally be included in the composition. Injection isrepeated once every three weeks for a total of four injections.Immunizing doses of the immunogenic composition are determined bymethods that will be appreciated by those having ordinary skill in theart. Stimulation of an immune response in the patient is monitored bystandard techniques, such as ELISA. Serum isolated from a blood sampletwelve weeks after the first injection of the immunogenic composition isfound to contain a high concentration of antibodies reactive with C.parvum as detected by indirect immunofluorescence staining. In contrast,a serum sample obtained from the same patient prior to the firstinjection of the immunogenic composition showed substantially noantibodies reactive with C. parvum. This indicates that the patient hasdeveloped an immune response against C. parvum as a result of havingbeen administered with an immunogenic composition comprising thepurified CSL high molecular weight glycoprotein antigen reactive withmAb 3E2. The same patient does not develop diarrhea when challenged withlive C. parvum administered orally. This latter observation proves thatimmunization with a composition comprising the purified CSL highmolecular weight glycoprotein antigen reactive with mAb 3E2 confersprotective immunity against infection with C. parvum.

What is claimed is:
 1. An isolated antigen of Cryptosporidium parvumcomprising a glycoprotein of approximately 1,400 kDa, as measured bysodium dodecyl sulfate polyacrylamide gel electrophoresis, said antigenhaving disposed thereon an epitope specifically recognizable bymonoclonal antibody 3E2, secreted by a hybridoma deposited as accessionnumber ATCC HB 12075, wherein the antigen is present in unisolated formin dense granules in sporozoites.
 2. An immunogenic compositioncomprising the antigen of claim
 1. 3. The immunogenic composition ofclaim 2, further comprising an adjuvant.
 4. An immunogenic compositioncomprising a substantially purified Cryptosporidium parvum antigencomprising a glycoprotein of approximately 1,400 kDa, as measured bysodium dodecyl sulfate polyacrylamide gel electrophoresis, wherein saidantigen is specifically recognizable by monoclonal antibody 3E2,secreted by a hybridoma deposited as accession number ATCC HB 12075, ina pharmaceutically acceptable carrier.
 5. The immunogenic composition ofclaim 4, further comprising an adjuvant.
 6. A method of stimulating ananti-Cryptosporidium parvum immune response in a mammal comprisingadministering to the mammal an immunogenic composition comprising asubstantially purified Cryptosporidium parvum antigen specificallyrecognizable by monoclonal antibody 3E2, secreted by a hybridomadeposited as accession number ATCC HB 12075, in a pharmaceuticallyacceptable carrier, wherein said antigen is a glycoprotein ofapproximately 1,400 kDa, as measured by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
 7. The method of claim 6, whereinsaid stimulating produces an immune serum or colostrum comprisingantibodies having an epitope specificity of the monoclonal antibody 3E2,accession number ATCC HB
 12075. 8. The method of claim 7, comprisingpassively immunizing a second mammal of a same or different species withthe immune serum or colostrum.
 9. The method of claim 8, wherein thesecond mammal is a human being.